Human Immunodeficiency Virus (HIV) is a retrovirus that infects and invades cells of the immune system and causes acquired immunodeficiency syndrome (AIDS). HIV breaks down the body's immune system and renders the patient susceptible to life-threatening opportunistic infections and cancers. The immune defect appears to be progressive and irreversible, with a high mortality rate that approaches 100% over several years.
The management of HIV/AIDS normally includes the use of multiple antiretroviral drugs in an attempt to control HIV infection. Antiretroviral therapy (ART) includes several classes of antiretroviral agents that act on different stages of the HIV life-cycle: (a) entry inhibitors interfere with binding, fusion and entry of HIV-1 to the host cell by blocking one of several targets; (b) nucleoside and nucleotide reverse transcriptase inhibitors (NRTI and NTRTI) are nucleoside and nucleotide analogues which inhibit reverse transcription; (c) non-nucleoside reverse transcriptase inhibitors (NNRTI) inhibit reverse transcriptase; (d) integrase nuclear strand transfer inhibitors (INSTI) inhibit the viral enzyme integrase, which is responsible for integration of viral DNA into the DNA of the infected cell; and (e) protease inhibitors (PI) block the viral protease enzyme necessary to produce mature virions upon budding from the host membrane (www.aidsinfo.nih.gov/guidelines).
The use of multiple drugs that act on different viral targets is known as highly active antiretroviral therapy (HAART). HAART was introduced in 1996 and involves various combinations of HIV inhibitors including, but not limited to, NRTI, NNRTI and PI (Schmit et al., The Journal of Infectious Diseases, 1996, 174 (5): 962-8). HAART has been effective in reducing mortality and progression of HIV-1 to AIDS and in improving patient quality of life.
Although ART has decreased morbidity and mortality for HIV, long-term therapy is associated with toxicity (Gardner et al., Toxicol Pathol, 2013—Review). As ART use increases worldwide, the need for toxicity monitoring has become imperative. The 2013 UNAIDS report on the global AIDS epidemic highlights ART pharmacovigilance as a priority for HIV care programs. The prevalence of all ART adverse events is reported to be 47% for clinical and 27% for laboratory manifestations, with 9% to 16% of these leading to changes in medication, hospitalization, chronic disability, or death (Fellay et al., Lancet, 2001, 358:1322-1327). Prevalence data for ART toxicity in children are scarce, although one study of 3936 HIV-infected children <5 years old living in low resource settings found that 3.8% had to switch to a second-line drug regimen within the first 36 months of ART due to medication toxicity (Sauvageot et al., Pediatrics, 2010, 125:e1039-1047).
Mitochondria organelles are found in every cell of the human body except red blood cells and play a major role in energy production and glucose and fat metabolism. Early effects of mitochondrial toxicity include a decrease in energy production and an increased production of lactate. Mitochondrial toxicity has been suggested as a common pathway for a variety of nucleoside-related adverse effects, including asymptomatic hyperlactatemia or varying degrees of fatigue, shortness of breath, nausea, vomiting, abdominal pain, weight loss, and even severe fatal lactic acidosis with severe liver steatosis. Also, cardiomyopathy, peripheral neuropathy, pancreatitis, hepatotoxicity, lipoatrophy and lipodystrophy have been attributed to underlying nucleoside-related mitochondrial toxicity (Birkus et al., Antimicrob Agents Chemother, 2002, 46(3):716-23; Fellay et al., Lancet, 2001, 358:1322-1327 and Renner et al., J Int AIDS Soc 2013, 16:18024).
In general, clinical manifestations of ART-induced mitochondrial toxicity can affect nearly any organ system (Gardner et al., Toxicol Pathol, 2013—Review). The underlying mechanism for mitochondrial toxicity, in the case of nucleoside reverse transcriptase inhibitors (NRTI), is attributed to inhibition of polymerase gamma, the key enzyme responsible for mitochondrial DNA replication (Johnson et al. J Biol Chem. 2001, 276:40847-57; Martin et al., Antimicrob Agents Chemother, 1994; 38:2743-9; Kohler et al., Environ Mol Mutagen, 2007, 48:166-172). However, NRTI, as well as other classes of antiretroviral agents such as non-nucleoside reverse transcriptase inhibitors and protease inhibitors, have also been reported to increase rates of apoptosis (Braga et al., BMC Gastroenterol 2010, 10:90), whether as a result of or in addition to mitochondrial damage is not certain. Outside of clearly defined clinical manifestations, diagnosis of mitochondrial toxicity is difficult given that there is no gold standard test. A combination of clinical symptoms, laboratory testing, and imaging studies, culminating in tissue biopsy to look for mitochondrial damage is deemed to be the most accurate method (Haas et al., Pediatrics 2007, 120:1326-1333), though it is cost-prohibitive and invasive. Thus, a “trial and error” approach to ART toxicity, in which medications are stopped or switched to see if the clinical manifestations resolve, has become commonplace. This practice favors the emergence of drug-resistant strains of HIV (Arnedo-Valero et al., Clin Infect Dis 2005, 41:883-890) and could lead to inappropriate use of second-line medications, especially in resource-limited settings.
Cytochrome C (Cyt-C) is a protein made from nuclear DNA as apocytochrome c, which is then shuttled across the mitochondrial outer membrane and transformed to a heme-containing, water soluble protein of approximately 12 kDa (Kulikov, et al., Cell Mol Life Sci, 2012, 69(11): p. 1787-97). It acts as a substrate in oxidative phosphorylation. Its release into the cytosol of the cell is a pro-apoptotic signal (Ow et al., Nat Rev Mol Cell Biol, 2008, 9(7): p. 532-42). In vivo, Cyt-C has been detected extracellularly during pro-apoptotic states (Renz A et al., Blood, 2001, 98:1542-1548). It has also been identified in circulation in experimental models of drug induced renal and hepatic toxicity (Small et al., Expert Opin Drug Metab Toxicol, 2012, 8:655-664; Miller et al., J Appl Toxicol, 2008, 28:815-828). Cyt-C can be detected in serum and/or plasma using a commercial enzyme linked immunosorbant assay (ELISA) (Marenzi et al., Am J Cardiol, 2010, 106(10): p. 1443-9; Dincer et al., Clin Invest Med, 2009, 32(4): p. E266-70; Barczyk et al., Int J Cancer, 2005, 116(2): p. 167-′73; Dincer et al., J Thromb Thrombolysis, 2010, 29(1): p. 41-5).
There is a need in the art for methods for detecting and quantifying ART-induced mitochondrial toxicity. Furthermore, there is a need in the art for a non-invasive, cost-effective diagnostic test with strong sensitivity and selectivity so as to inform clinicians on the status of the patient undergoing ART, in order to prevent unnecessary interruptions in ART, guide the use of second-line regimens and thus provide the best treatment modalities. The present invention satisfies these needs.